The overall goal of this IPCAVD proposal is the development and clinical testing of an Envelope immunogen optimized to elicit broadly neutralizing anti-HIV-1 antibodies. The goal of Project 2 is the preclinical evaluation of HIV-1 Envelope vaccine immunogens that have been optimized to engage the predicted germline precursor B cell receptors of neutralizing antibodies that target the CD4 receptor binding site and begin the process of their development. We hypothesize that one key reason for the failure of previous generations of Envelope immunogens to elicit such antibodies is that these immunogens do not engage germline B cell receptors, the key precursors to broadly neutralizing 'VRCOI '-class antibodies. Thus, previous immunogens did not efficiently initiate and drive the process of 'VRCOI'-class bNAb antibody maturation. In this project we will evaluate germline optimized Env immunogens for their ability to effectively initiate the development of broadly neutralizing anti-CD4-binding site antibodies of the 'VRCOI'-class, and will optimize the vaccine strategies that will be deployed in human clinical trials in Project 4. Utilizing four complementary animal model systems: lentigenic mice, transgenic mice , humanized Veloclmmune mice, and non-human primates, we will evaluate the ability of our immunogens to stimulate germline VH genes known to be the progenitors of 'VRCOr class broadly neutralizing antibodies, trace their maturation over the course of vaccination, and evaluate their ability to block HIV-1 infection. Ultimately, we will determine which optimized Envelope immunogen, and which vaccine modality is the most appropriate for deployment into human clinical trials. A second goal of this Project 2 is to confirm that similar engagement and maturation of germline BCRs that give rise to 'VRC01' class antibodies also takes place in humans immunized with our immunogens. Our studies will provide invaluable information on how well such broadly neutralizing antibodies can be stimulated by germline-optimized Envelope immunogens and how far we are able to drive antibody maturation by vaccination. Such a detailed analysis of vaccine-elicited anti-HIV-1 responses will provide valuable insight for future immunogen and vaccine regimen design efforts.